1. Field of the Tnvention
The present invention relates to detoxified and immunologically active proteins (“mutant LTs”), more specifically, to mutant LTs having mutated amino acid sequences of heat-labile enterotoxin of E. coli, DNA sequences encoding the mutant LTs, recombinant expression vectors comprising the DNAs, recombinant microorganisms transformed with the recombinant expression vectors, process for preparing the mutant LTs and pharmaceutical application of the said proteins as immunogenic antigens for vaccination and as adjuvants for antibody production.
2. Description of the Prior Art
Enterotoxigenic Escherichia coli (“ETEC”) strain causes diarrheal disease in humans and animals due to production of toxin such as heat-labile enterotoxin (“LT”) (see: Spangler, B. D., Microbiol. Rev., 56:622-647(1992)). LT is a multimeric protein composed of two functionally distinct domains: an enzymatically active A subunit (“LTA”) of ˜30,000 daltons with ADP-ribosylating activity, and a pentameric B subunit (“LTB”) of ˜11,600 daltons that contains GM1 (momosialoganglioside) receptor-binding site (see: Bäckström, M. et al., Mol. Microbiol., 24:489-497(1997)). Upon thiol reduction, the A subunit dissociates into two polypeptide chains, i.e., A1 (Mr, 23,000 daltons) and A2 (Mr, 6,000 daltons) (see: Tsuji, T. et al., J. Biol. Chem., 260:8552-8558(1985); Grant C. C. R. et al., Infect. Immun., 62:4270-4278(1994)). The A1 subunit, in particular, intoxicates eucaryotic cells by catalyzing ADP-ribosylation of Gs, a GTP-binding protein that regulates the levels of the second messenger cAMP (see: Guerrant, R. L. et al., Infect. Immun., 10:320-327(1974); Field, M. et al., N. Engl. J. Med., 321:800-806(1989)). The resulting increase in cAMP level causes secretion of water and electrolytes into the small intestine through interaction with two cAMP-sensitive ion transport mechanisms including (i) NaCl co-transport across the brush border of villous epithelial cells and (ii) electrogenic Na+-dependent Cl− secretion by crypt cells (see: Guidry, J. J. et al., Infect. Immun., 65:4943-4950(1997)).
Both the cholera toxin (“CT”) from Vibrio cholerae and heat-labile enterotoxin from ETEC belong to the most potent mucosal adjuvants and immunogens known to date by oral and other mucosal routes, via which most of antigens are unable to induce immune responses (see: Jackson, R. J. et al., Infect. Immun., 61:4272-4279(1993); Takahashi, I. et al., J. Infect. Dis., 173:627-635(1996)). However, their toxicities have precluded their clinical use in humans (see: Douce, G. et al., Proc. Natl. Acad. Sci., USA, 92:1644-1648(1995)). One approach to overcome the problem of toxicity is the generation of genetically detoxified derivatives of LT (see: Lobet, Y. et al., Infect. Immun., 59:2870-2879(1991); Dickson, B. L. and Clements, J. D., Infect. Immun., 63:1617-1623(1995)) and CT (see: Fontana, M. R. et al., Infect. Immun., 63:2356-2360(1995); Yamamoto, S. et al., Proc. Natl. Acad. Sci., USA, 94:5267-5272(1997b)) by site-directed mutagenesis of amino acids which are located on the β-strand that constitutes the ‘floor’ of NAD-binding cavity.
The most important factor for immunogenicity is shown to be the ability to bind to the receptor on eucaryotic cell (see: Nashar, T. O. et al., Proc. Natl. Acad. Sci., USA, 93:226-230(1996)). In fact, a non-binding mutant of the B subunit of LT was found to be non-immunogenic (see: Guidry, J. J. et al., Infect. Immun., 65:4943-4950(1997)). In addition, another group found that the ADP-ribosylating activity is unnecessary for immunogenicity because nontoxic derivatives of LT obtained by site-directed mutagenesis of the A subunit retained the immunological properties of the wild-type LT (see: Pizza, M. et al., J. Exp. Med., 180:2147-2153(1994)).
The attempt to define the role of ADP-ribosylating activity in adjuvanticity of LT has generated conflicting results. For example, it was reported that a nontoxic derivative of LT (LTE112K) when co-administered with keyhole limpet hemocyanin (KLH) by an oral route in mice, lacked the adjuvant properties, thus suggesting that the adjuvanticity of LT is linked to its ADP-ribosylating activity (see: Lycke, N. et al., Eur. J. Immunol., 22:2277-2281(1992)). However, more recently, the adjuvant activity of the LTE112K was found to be identical to that of the LT holotoxin when delivered with influenza virus surface antigen by an intranasal route (see: Verweij, W. R et al., Vaccine, 16:2069-2076(1998)). On the other hand, other investigators showed that another LT derivatives, LTK63, lacking enzymatic activity and toxicity was still able to elicit antibody responses against the co-administered antigen in mice immunized orally, intranasally, or intravaginally (see: Di Tommaso, A. et al., Infect. Immun., 64:974-979(1996); Giuliani, M. M. et al., J. Exp. Med., 187:1123-1132(1998); Marchetti, M. et al., Vaccine, 16:33-37(1998)).
Under the circumstances, the present inventors, based on the findings that detoxified LT derivatives may induce antibody responses, tried to explore an efficient immunogenic antigen and mucosal adjuvant for vaccination, which can be applied for the development of a mucosal vaccine as well as a novel diarrheal vaccine for humans and animals.